EP1474234A1 - Enantioselective cation-exchange materials - Google Patents

Abstract

The invention relates to a enantioselective cation-exchange material comprising a chiral selector (1) consisting of a chiral component (2) and at least one cation-exchange group (X), a spacer (3), and a carrier (4). The cation-exchange material is characterized by the fact that the chiral component (2) has a molecular weight of less than 1,000 while the at least one cation-exchange group (X) is an acid group having a pKa<4.0.

Description

 Enantio selective cation exchange materials

The present invention relates to enantioselective cation exchange materials, comprising a chiral selector, which is composed of a chiral component and at least one cation exchange group, a spacer and a support.

In particular, the present invention relates to enantioselective molecule recognition materials of the cation pairing type and of the cation exchange type, which represent a new class of separating materials which, for specific interaction with complementarily structured molecules on their surface, have new chiral selector components with a free functional acid group or with free functional groups Wear acid groups. In addition, their use in molecular recognition concepts for highly selective binding and separation, isolation and purification of basic chiral compounds including chiral synthons, drugs, amino acids, peptides, proteins, aminoglycosides and other basic chiral compounds is the subject of this invention.

Background of the Invention

Biological systems consist of inherently chiral components, e.g. Proteins that can interact differently with the stereoisomers of endogenous and exogenous chiral compounds. As a result, stereoisomers of exogenous compounds such as drugs, toxins, agrochemicals or food additives very often have different pharmacological and toxicological profiles. This effect, for example, forced drug developers to study the pharmacological profiles of the individual enantiomers separately, and also led to an increased number of drugs that are manufactured and marketed as a single enantiomer. A prerequisite for this is the availability of preparative processes for enantiomer production and analytical tools for stereoselective analysis of the quality and the pharmacokinetic effects.

Individual enantiomers can be prepared by stereoselective synthesis or racemate resolution. The latter approach is often the preferred and more widely available method, since syntheses of racemates are usually easy and inexpensive to perform compared to stereoselective synthesis Racemate splitting methodologies are often applicable to different, structurally different classes of compounds. The latter can also be the preferred approach when both enantiomers are used in a purely enantiomeric form, such as for pharmacological experiments. In any case, the latter approach requires chiral selectors or functional materials modified with selector groups that can interact stereoselectively with the two enantiomers when contacted with the racemate.

The present invention now discloses such new chiral selectors and novel functional materials which can be used in various preparative racemate resolution or enantiomeric separation concepts including chromatographic solid-liquid or liquid-liquid processes, solid-liquid or liquid-liquid extraction technologies and membrane separation techniques can be applied. Equally, the chiral selectors and functional materials can also be used in analytical enantiomer separation processes, where they are integrated as adsorption materials in columnar liquid chromatography, supercritical liquid chromatography, capillary electrochromatography, in chip technologies or as molecular recognition materials and sensitive layers in sensor technologies.

The enantioselective cation exchange material according to the invention comprises a chiral selector, which is composed of a chiral component and at least one cation exchange group, a spacer and a support and is characterized in that the chiral component has a molecular weight of less than 1,000 and the at least one cation exchange group has an acid group a pKa <4.0.

The enantioselective cation exchange material is preferably characterized in that the acid group has a pKa <3.5. It is further preferred that the acid group has a pKa <2.5.

In a further preferred embodiment, the enantioselective cation exchange material according to the invention is characterized in that the acid group is a sulfone, sulfine, phosphorus, phosphone or phosphine group.

The present invention is based on the surprising finding that the chiral recognition capabilities of enantioselective cation exchangers improve with increasing acidity of the synthetic chiral selector of low molecular weight. The primary target compounds of the present invented enantioselective molecular recognition materials of the cation pairing and cation exchange type are basic chiral drugs and intermediates (chiral synthons). Since a high percentage of the total market share of chiral drugs contains basic functional groups, which makes them accessible for separation by the invented functional molecular recognition materials, these should have a wide range of applications.

In addition, many biomolecules are also chiral, containing basic functionalities, and thus the functionalized solid phases or materials of the present invention are also useful for separations of basic biomolecules, especially of closely related analogues and isomeric forms. Areas of application of the novel materials therefore include the separation of amino acids, peptides, proteins, nucleotides, aminoglycosides and many other basic chiral compounds.

The most important features of the present invention are briefly described below. The present invention relates to enantioselective molecular recognition materials of the cation exchange type and cation pairing type, which are composed of at least 3 modules, which i) an acidic chiral selector, which is composed of a chiral component or a building block with a functional acid group, ii) a spacer and iii ) are a support or polymer backbone according to the general scheme shown in FIG. 1.

In any case, the invented functional materials have at least 1 free functional acid group (hereinafter referred to as the X substituent). The X substituent is a strongly acidic functional group with a pK _a <4.0 (based on a completely aqueous system; for the conditions of the pK _a determination see Example 7) like a sulfone, sulfine, phosphorus, phosphone -, phosphinic, boronic, amidophosphonic, amidosulfonic acid or any other acid group. From a molecular recognition point of view, this functional acid group controls the interaction with the cationic target, the analyte or the sample components through strong intermolecular ionic interactions according to ion pairing and ion exchange mechanisms, which are stronger the lower the dielectric constant of the medium, but also in aqueous ones or aqueous-organic liquid phases are active. It was found that with decreasing pK _{a of} the cation exchange group, ie with increasing acidity of the functional acid group of the selector, the strength of the selector analyte binding increases. Surprisingly, stronger binding also improved the ability to bind and separate enantioselectively. This was far from expected: Although a stronger cation exchanger can be expected to bind oppositely charged analytes more strongly than a weak counterpart, higher enantioselectivity could not be expected as a logical consequence. In contrast, experience has shown that analytes that are too strongly bound very often result in only moderate enantioselectivity.

The chiral component bearing the acidic X substituent is a chiral compound in a single enantiomeric form or is constructed from enantiomerically pure chiral synthons. Both together, the chiral component and the X substituent, form the chiral acidic selector of low molecular weight, which forms the core of the functional materials at hand, since they provide the information for selective molecular recognition of the target compounds. In contrast to macromolecular structures such as proteins, the chiral component preferably has a low molecular weight and is typically composed of chiral synthons such as natural or unnatural, cyclic or non-cyclic amino acids, hydroxycarboxylic acids, aminophosphonic acids, aminophosphinic acids, aminosulfonic acids, aminosulfinic acids, aminoboronic acids, hydroxyphosphonic acids, mercaptophonic acids , Tartaric acid derivatives, mandelic acid derivatives, camphorsulfonic acid derivatives, linear or cyclic, natural and non-natural peptides, linear or cyclic sulfopeptides, linear or cyclic phosphonopeptides. The low molecular weight chiral acidic selector can also be an amphoteric compound that is negatively charged when used in conditions below the isoelectric point. In addition to the primary ionic interaction site created by the X substituent, optimally functioning variants of molecular recognition materials of the cation exchange and cation pairing type also have other interaction sites such as hydrogen donor acceptor groups (denoted by Y) and / or π-π interaction sites (aromatic groups with preferably electron-withdrawing or electron-donating substituents) and bulky elements for repulsive and / or attractive interactions of the van der Waals type. The functional Y substituent can be an amide, carbamate, sulfonamide, urea, carbonyl, semicarbazide, hydrazide or sulfonimide group or other similar hydrogen donor acceptor system. Bulky elements of the selector component and interacting functional groups are composed such that a binding pocket is preformed, the polar interaction sites being located more in the center of the pocket and the hydrophobic bulky groups on the edge of the pocket. Target analytes that meet the binding requirements sterically and electrostatically can bind specifically to this pocket, while others are excluded from strong binding.

For many applications, these selectors must be immobilized on solid or occasionally also on liquid matrices, which are called carriers, in such a way that the selector groups are correctly exposed to the surrounding solution, which contains the target compounds to be recognized and bound, thereby enabling their binding , The carrier should be inert to the binding of the target compound, but has the function of guaranteeing the chemical and physical stability of the molecular recognition material. In flow applications such as chromatography, the carrier or its physical properties determine the kinetic properties of the materials. It is therefore an important part of the functional material. In the present invention, the carrier can be an inorganic, organic or mixed inorganic-organic hybrid-like material. Such carrier materials include commercially available and self-developed beads, monolithic or continuous materials, nanoparticles, membranes, resins, surface-limited layers, which are made from chemical materials including silicon dioxide (SiO ₂ ), aluminum oxide (Al ₂ O ₃ ), zirconium dioxide (ZrO ₂ ), Titanium oxide (TiO ₂ ), materials derived from sol-gel, organic-inorganic siliceous hybrid materials, optionally crosslinked polysiloxanes, any polymer obtained from vinyl monomers, optionally crosslinked poly (meth) acrylates, optionally crosslinked poly (meth) acrylamides, optionally crosslinked polystyrenes, mixed Styrene (meth) acrylate polymers, ring opening methathesis polymers, polysaccharides, agarose, and any of these materials that have been specifically functionalized to allow immobilization of the low molecular weight chiral acid selector. Among the preferred carriers are silica beads, poly (meth) acrylate polymer beads, poly (meth) acrylamide beads, poly (meth) acrylate monoliths, polystyrene resins, which are optionally modified with attached reactive groups to immobilize the selector.

A polymer backbone or a polymer matrix can also be regarded as a carrier, so that a spacer and chiral acidic selector represent actually attached chiral groups of the polymer which are responsible for targeted molecular recognition of the basic target compounds. The main function of the spacer is to bind the low molecular weight acidic selector to the support. Both the length and the chemical functionality of the spacer are variable. To a certain extent he can participate in the selector-analyte binding with a positive but also a negative effect on the selectivity. It can influence the rigidity and accessibility of the selector and also have an effect on the binding characteristics. Ultimately, it determines the chemical stability of the functional materials and can influence their compatibility with analytes, a factor that is particularly important for biomolecule separation. All general solid phase linker concepts and approaches for the production of chromatographic stationary phases can be used for the synthesis of the present functional materials. Immobilization strategies that are preferred for the synthesis of currently invented chiral cation exchange materials include the reaction of a vinyl-modified acidic selector with a thiol-modified carrier, in particular with thiol-propyl-modified silicon dioxide, by means of a radical addition reaction. Other immobilization concepts that can be used include the asymmetric reaction of a diisocyanate linker with an amino- or hydroxyalkyl-modified carrier and an amino- or hydroxy-modified selector component, the reaction of an amino-, hydroxy- or thiol-modified carrier with a chlorine- or bromalkanoyl-derivatized selector, the reaction of alkoxy- or chloroorganosilane with a terminal, reactive functionality for coupling to a selector component, the hydrosilylation reaction of alkoxy- or chlorohydrosilane with a selector containing a vinyl group, the coupling of an amino-modified carrier and an amino-modified selector by reaction of one of the two components with a dicarboxylic acid anhydride spacer component and the subsequent activation of the resulting carboxylic acid function and reaction with a second amino component and many other immobilization strategies, which generally one can be used to immobilize chiral selectors, proteins and peptides on solid supports.

In special cases, the current enantioselective type of separation of the cation exchange type can be combined with other types of separation such as reverse phase chromatography, etc. For example, by using new surface modifications, which are obtained by combining the above-described selector components with long-chain alkyl groups, which leads to RP-chiral mixed-form selectors of the cation exchange type and solid phases with dedicated selectivity and separation character. One of the particular advantages is that the functional materials described above are resistant to more or less all normally used liquid phase systems. Thus, these functional materials can be operated with manifestations of the aqueous, aqueous-organic (reverse phase), polar organic phase and apolar organic phase (normal phase), the liquid or mobile phase for operation in the ion exchange mode in any case a cation, preferably an ammonium or an organic ammonium compound as a counter ion.

The present invention also relates to the use of all processes which use the novel molecular recognition materials of the cation exchange type and cation pairing type in preparative chromatographic solid-liquid or liquid-liquid processes, solid-liquid or liquid-liquid extraction technologies and membrane separation techniques. In analogy to this, their application in analytical methodologies, etc. integrated as adsorption materials in column liquid chromatography, supercritical liquid chromatography, capillary electrochromatography, in chip technologies or as molecular recognition materials and sensitive layers in sensor technologies, likewise the subject of the present patent application.

State of the art

A variety of chirally functionalized adsorption materials have previously been developed for the purpose of enantiomer separation. A detailed overview of enantioselective stationary phases and the corresponding processes which use such materials for HPLC enantiomer separation is given elsewhere [M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: R.M. Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437]. Some of the functionalized chiral materials associated with the present invention in one aspect or another are described in the following section.

Chiral low molecular weight ion exchangers:

The inventors of the present invention previously disclosed the discovery of chiral separation materials of the anion exchange type which have synthetic basic chiral selectors of low molecular weight immobilized on solid supports and which were used to separate stereoisomers from acid compounds (Cinchonan Based Chiral Selectors for Separation of Stereoisomers. Lindner, W ., Lämmerhofer, M., Maier, NM, PCT Int. Appl. (1997) WO 9746557, EP 912563, US 6313247). Detailed descriptions of this work were later published in various papers [M. Lämmerhofer et al., J. Chromatogr. A, 741 (1996) 33; A. Mandl et al., J. Chromatogr. A, 858 (1999) 1; NM Maier et al, Chiraiity, 11 (1999) 522], see also chapter 9.2.3.2. from [M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: RM Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437] and references contained therein. The same selectors and chiral stationary phases were also used in capillary electrochromatography (CEC) [M. Lämmerhofer and W. Lindner, J. Chromatogr. A, 829 (1998) 115-125] or were specially adapted for their use in the CEC [M. Lämmerhofer et al., Anal. Chem., 72 (2000) 4614-4628; J.MJ. Frechet, F. Svec, M. Lämmerhofer, Electrochromatographic device for use in enantioselective Separation and enantioselective Separation medium for use therein. US Patent Application Ser.No. 09 / 645,079 (2000).]. Related chiral stationary phases based on Ergot alkaloid selectors were developed by M. Flieger and M. Sinibaldi and co-workers, see references 392-395 by [M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: RM Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437]. All of these alkaloid-derived chiral materials are conceptually related to the present invention through the existence of enantioselective chiral ion exchange mechanisms that involve ionic interactions between oppositely charged chiral selectors and analytes. However, this is an approach that is reciprocal to what is claimed in the present patent application, which means that a single enantiomer of a cationic chiral compound is used as a chiral selector and is immobilized on solid supports or incorporated into polymeric frameworks, what leads to chiral anion exchangers. The range of applicability of such enantioselective anion exchangers is of course more or less limited to the separation of chiral acids. In theory, using the reciprocity principle of chiral recognition [W. Pirkle et al., J. Chromatogr., 192 (1980) 143-158; W. Pirkle and R. Däppen, J. Chromatogr., 404 (1987) 107-115], immobilizing a single enantiomer of the acidic binding partner of the chiral anion exchange concepts described above, developing enantioselective stationary phases which separate the enantiomers from chiral ones Bases would allow. In practice, however, this principle can easily fail, as described below, and its experimental implementation is not trivial. At this point it should be emphasized that the reciprocity principle of chiral recognition was originally for stationary phases were discovered by the Pirkle concept (vide supra), which also uses neutral selectors and analytes, and it was therefore unclear whether it also applies to the chiral ion exchange concept with charged selectors and analyte species.

Encouraged by the success of the chiral anion exchange approach, the inventors tried to extend this concept reciprocally to basic analytes, which led to chiral cation exchangers of low molecular weight, unfortunately with very negative results [E. Veigl et al., J. Chromatogr. A, 694 (1995) 151]. In contrast to the successful examples reported in the Pirkle concept, the reciprocity principle of chiral recognition did not work, to the surprise of enantiomeric ion exchangers. Similar problems in the implementation of the reciprocity principle have already been reported in several publications, which also dealt with chiral stationary phases from the Pirkle concept [M.H. Hyun et al., J. Chromatogr. A, 922 (2001) 119-125].

The work of Veigl et al. was the only attempt at enantioselective cation exchangers reported so far, but the materials developed were completely useless. These completely negative results were in a in Chromatogr. published treatise described and interpreted [E. Veigl at al., J. Chromatogr. A, 694 (1995) 151] and led among the scientists working in the field of chiral separation to the technical prejudice that such enantioselective cation exchangers can function successfully for no reason. This is also reflected in the long time that passed before the inventors were able to surprisingly overcome this technical prejudice. The key to the success of the present invention was the discovery that the chiral recognition capabilities of enantioselective cation exchangers improve with increasing acidity of the synthetic chiral low molecular weight selector. This step of the invention has not been surpassed in the past, and the inexpensive, strongly acidic functional structural element has never been combined with other suitable structural elements (small distance between the stereogenic center and the primary ionic interaction site, which are either in the -, ß- or γ-position to the stereogenic center , steric barriers, aromatic groups for a π-π interaction), which are necessary for successful chiral selectors. The novel enantioselective cation exchangers, which combine all the favorable structural features mentioned, showed extremely promising results with regard to the enantioselective molecular recognition and enantiomer separation of chiral bases of different types. Preferred are the strong enantioselective cation exchangers based on sulfonic or phosphonic acid groups 10

supporting selectors are based on their large enantio-recognition abilities to clearly distinguish them from the unsuccessful precedents of the above treatise (see examples).

Chiral ion pair chromatography:

In general terms, chiral ion pair chromatography can be considered somehow related to the methods of enantioselective ion exchange chromatography [reviewed by C. Pettersson and E. Heldin in: A practical approach to chiral separations by liquid chromatography. G. Subramanian (ed.), VCH, Weinheim, 1994, pp. 279-310]. For example, for the separation of chiral bases, acidic chiral counterions such as 2,3,4,6-di-O-isopropylidene-2-keto-L-gulonic acid, Z-derivatized amino acids, di- or tripeptides, 10-camphorsulfonic acid, tartaric acid and Tartaric acid derivatives used as additives to the mobile phase [see C. Pettersson and E. Heldin in: A practical approach to chiral separations by liquid chromatography. G. Subramanian (ed.), VCH, Weinheim, 1994, pp. 279-310, and references contained therein]. These chiral counterions form stereoselectively electrically neutral ion pairs with chiral bases in the mobile phase, which have different adsorption properties on achiral stationary phases and stationary phases of the reverse phase type. As a secondary equilibrium, the chiral counterion can also be adsorbed onto the stationary phase and thus forms a dynamically coated chiral ion exchanger. The disadvantage of such an approach is the presence of the selector in the flowing medium, which causes disturbances and problems with most detection methods and prevents their use in preparative concepts. Indeed, the covalent adherence of the selector to the stationary phase in the materials of the invention and the resulting elimination of the selector from the effluent is a considerable advantage over the mode of ion-pair chromatography.

Chiral ligand exchange separation materials:

It could be assumed that chiral ligand exchange type stationary phases [reviewed by VA Davankov, J. Chromatogr. A, 666 (1994) 55; and by A. Kurganov, J. Chromatogr. A, 906 (2001) 51-71; and by M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: RM Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437], who use stereoselective chelation as the separation principle, somehow with the above described chiral ion exchanger of low molecular weight. Amino acids, in particular proline derivatives, are most often immobilized dynamically or covalently as chelating agents or chiral selectors on suitable supports, which form selectively mixed metal complexes with analytes, which must also have chelating properties for the metal ions dissolved in the liquid (mobile) phase. There are generally at least 2 distinct differences when compared to the materials of the present invention: i) Instead of an ion exchange type selector, a chelator is immobilized on the support. Such chelators are often amino acids such as proline, hydroxyproline or penicillin derivatives, as mentioned above [regarding the structures, see above review article and references contained therein]. Functional materials of the chiral ligand exchange (CLEC) type usually have both acidic and basic functional groups, ii) Completely different molecular recognition and SO-SA binding mechanisms are involved. In contrast to the separation materials of the cation exchange type according to the invention, no ion-ion interactions are formed between acidic selector components and cationic groups of the analyte. For example, enantiomers of amino alcohols with phases of the CLEC type can be resolved, the functional acid group of the phase of the CLEC type with the positively charged metal ion, which, in the mixed selector-metal analyte complex, also acts as an electron acceptor for the basic function of the analyte. interacts.

Chiral crown ether phases

Recently Machida et al. a new chiral stationary crown ether phase [Y. Machida et al, J. Chromatogr. A, 805 (1998) 85-92; MH Hyun et al., J. Chromatogr. A, 822 (1998) 155-161] for the separation of the enantiomers of chiral primary amines. The crown ether component of this CSP comprises carboxylic acid groups. However, the primary amines are complexed by inclusion in the cavity of the macrocyclic crown and kept inside, in particular because of the simultaneous triple hydrogen bond between the NH of the ammonium ion and the ether oxygen, as has been shown by NMR experiments [Y. Machida et al., J. Chromatogr. A, 810 (1998) 33-41: p.39]. Steffeck, Zelechonok and Gahm in J. Chromatogr. A 947 (2002) 301-305 describe an enantioselective separation of racemic secondary amines on this chiral stationary liquid chromatography phase based on crown ether. Chiral stationary phases, based on macromolecular and medium-sized natural selectors;

Stationary phases of the protein type, e.g. based on Bovine Serum Albumin (BSA) [p. Alienmark et al., J. Chromatogr., 264 (1983) 63], human serum albumin (HSA) [E. Domenici et al., Chromatographia, 29 (1990) 170], cci-acidic glycoprotein (AGP) [J. Hermansson, J. Chromatogr. 269 (1983) 71], or Ovomucoid (OVM) [T. Miwa et al., J. Chromatogr., 408 (1987) 316], can be considered at a macromolecular selector level as counterparts to the currently invented low molecular weight selectors of the ion exchange type [for a more detailed list of proteins used for enantiomer separation see reviews by J. Haginaka, J. Chromatogr. A, 906 (2001) 253-273; and by M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: R.M. Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437; Chapter 9.2.1.2. Pp. 365-373]. Ionized functional groups of the side chains of the protein can be subjected to an ion-ion interaction with complementarily charged functional groups of the analytes. As highlighted, they differ from the materials of the present invention in the molecular size of the selector and the resulting unfavorable ratio of selective binding sites with high affinity per mole of protein and chiral stationary phase, resulting in poor loading capacity of the latter. In addition, protein selectors are completely natural selectors from a biological source formed purely from natural proteinogenic amino acids, and these distinguishing binding sites cannot be chemically tailored to better match the steric and functional preconditions of analytes. Dedicated derivatization and optimization strategies are impossible. Furthermore, they have a limited chemical and biological stability, disadvantages which do not exist in the materials according to the invention or which are overcome by using non-natural amino acids as building blocks.

Another, but different, macromolecular natural chiral selector that has been used to make chiral separation materials is heparin, a glucosaminoglycan that is a heterogeneous mixture of variably sulfated polysaccharide chains made up of repeating units of D-glucosamine and either L-iduron or D-glucuronic acid exist. Chloroquine enantiomers were separated using high-performance liquid chromatography using a chiral stationary phase based on heparin [A. Stalcup et al., Anal. Chem., 68 (1996) 13

13]. The heparin-based CSP is not very well characterized because it is randomly composed of varying repeating units (vide infra), and it also lacks important structural elements of the currently invented selectors such as steric barriers and π-π interaction sites.

Another group of natural source selectors are the macrocyclic antibiotics including vancomycin, teicoplanin, ristocetin and avoparcin [discussed by M. Lämmerhofer and W. Lindner, Recent developments in liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: R.M. Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437; Chapter 9.2.2.2., Pp. 381-392; and by T.J. Ward and A.B. Farris III, J. Chromatogr. A, 906 (2001) 73-89]. Chiral stationary phases have also been developed from these medium-sized macrocyclic selectors, which also have ionic interaction capabilities, e.g. from Vancomycin [D.W. Armstrong et al., Anal. Chem., 66 (1994) 1473], Teicoplanin [D.W. Armstrong et al., Chirality, 7 (1995) 474] and ristocetin [K.H. Ekborg-Ott et al., Chirality, 10 (1998) 434]. It has been shown that inclusion complex formation in the basket formed by the macrocyclic structure in combination with a multiple hydrogen bond on the peptide skeleton of the peptidomimetic are dominant as primary functional mechanisms, while interactions of the ion exchange type, if any, are of secondary importance. In contrast to the synthetic selectors of low molecular weight of the ion exchange type according to the invention, these natural selectors are not synthesized because of their complicated structure and are therefore not available in both enantiomeric forms.

Macromolecular synthetic chiral ion exchangers, obtained by a molecular imprinting process:

This class of ion exchangers includes some chiral separation media that are completely synthetic and are obtained by a matrix polymerization of ionizable functional monomers and complementarily ionizable template molecules, so-called molecularly printed polymers (MIP). If the template molecule is a single enantiomer of a chiral compound, chiral cavities can be formed, which can specifically bind the template again. Previously, the existence and predominance of a cation exchange retention mechanism for an acidic MIP, which was prepared from methacrylic acid as a functional monomer and basic phenylalanine anilide as a template, was established by Seilergren and Shea [B. Sellergren and KJ Shea, J. Chromatogr. A, 654 (1993) 17]. The most significant difference of these materials compared to those of the present invention is the (micro) heterogeneity of the Binding sites. As emphasized, the enantioselective binding sites of MIP type cation exchangers, in contrast to the enantioselective cation exchange materials according to the invention, where the binding site is fully specified and characterized, are composed of non-chiral functional monomers, which leads to poorly defined and heterogeneous binding sites. This adversely affects the performance of the release material, a problem inherent in the MIPs. More details can be found in a recently published review article [B. Sellergren, J. Chromatogr. A, 906 (2001) 227-252].

Chiral separation materials of the non-ion exchange type with some relationship to the present invention:

A large number of chiral stationary phases based on low molecular weight synthetic selectors use the same or similar chiral building blocks (chiral synthons) as disclosed in this application. The significant difference, however, is that the acid function is esterified or amidated, resulting in separation materials with neutral chiral selectors. Such CSPs are used for the separation of enantiomers of neutral chiral compounds or of basic and acidic chiral compounds under conditions in which the ionization is suppressed. This concept is known as the Pirkle concept; detailed lists of selectors and chiral stationary phases that have been developed and can be categorized as Pirkle concept phases are given in reviews of [M. Lämmerhofer and W. Lindner, Recent developments of liquid chromatography enantioseparation. In: Handbook of analytical separations. (Series ed .: RM Smith). Volume 1: Separation methods in drug synthesis and purification (Ed .: K. Valko) Elsevier, Amsterdam, 2000, pp. 337-437, chapter 9.2.3.1 .; Pp. 395-405; by F. Gasparrini et al., J. Chromatogr. A, 906 (2001) 35-40; and by C. Welch, J. Chromatogr. A, 666 (1994) 3]. Some of the most prominent representatives are phases based on N- (3,5-dinitrobenzoyl) phenylglycine (DNBPG) or N- (3,5-dinitrobenzoyl) leucine (DNBLeu) [WH Pirkle et al., J. Chromatogr., 348 ( 1985) 89; and WH Pirkle and JE McCune, J. Chromatogr., 479 (1989) 419], N-DNB-3-amino-3-phenyl-2-tert-butyl-propanoic acid (β-Gem 1) [WH Pirkle and JE McCune, J. Chromatogr., 441 (1988) 311], N- (2-naphthyl) alanine (NAP-Al) [WH Pirkle et al., J. Org. Chem., 51 (1986) 4991], N- (3,5-Dinitrobenzoyl) tyrosine (DNB Tyr-E and ChyRoSine) [N. Bargmann-Leyder et al., Chromatographia, 39 (1994) 673 and N. Bargmann-Leyder et al., Anal. Chem., 67 (1995) 952], N - {[l- (l-naphthyl) ethyl] amido} -tert.-leucine (Sumichiral OA-4600) [N. Oi et al., J. Chromatogr. A, 694 (1995) 129], N-DNB-α-amino-2,2-dimethyl-4-pentylphosphonic acid (α-Burke 1) [WH Pirkle and JA Burke, J. Chromatogr., 557 (1991) , 173] and L-Val-L-Val-L-Val tripeptide [N. Oi et al., J. Chromatogr. A, 722 (1996) 229]. about - -

15

other peptide-based CSPs have been reported, e.g. via CSP from Val tripeptide and 1- (l-naphthyl) amine as chiral synthons accumulated around triazine [A. Iuliano et al., J. Chromatogr. A, 786 (1997) 355], or via CSPs for N- (2-naphthyl) alanine diethylamide, which is based on dipeptide selectors which have been derivatized N-terminally with DNB and developed by a combinatorial approach [CJ. Welch et al., Enantiomer, 3 (1998) 471]. Again, it must be emphasized that none of these approaches have any ion exchange mechanisms, which clearly distinguishes them from the present invention.

Other unrelated approaches and materials:

Conventional cation exchange type release materials have immobilized achiral functional acid components on supports and are therefore unrelated to the functional materials of the present invention. They can be used to separate chiral bases, but are not enantioselective, i.e. they cannot separate the enantiomers from chiral bases. Similarly, the peptide-like poly (aspartic acid) silica sorbent (PolyCAT A) made from optically inactive poly (succinimide) [PolyN A] [C.N. Ou et al., J. Chromatogr., 266 (1983) 197] for the non-enantioselective cation exchange chromatography of proteins. This poly (aspartic acid) material was also modified with achiral taurine in such a way that a sulfoethylaspartamide-modified stationary phase (polysulfoethyl A) was obtained. It was also used for non-enantioselective cation exchange chromatography. The functional groups of these agents represent indefinite, randomly polymerized, optically inactive ligand molecules and therefore clearly differ from the chiral acid selectors of low molecular weight according to the invention, a well-characterized structure and additional structural features (eg π-π interaction site) being required in order to to be enantioselective. In fact, the polyCAT A and polysulfoethyl A phases mentioned above are not enantioselective for chiral bases.

Other stationary phases modified with peptides are used in non-enantioselective affinity chromatography concepts. Examples of such materials include monoliths modified with synthetic peptides and used for immunoaffinity chromatography [GA Platonova, et al., J Chromatogr. A, 1999, 852, 129-140], namely with a Ser-Pro-Gly-Phe-Arg sequence, which is synthesized directly on monolith disks and granular materials, which - after cleavage of the N-terminal protective group - then directly were used for immunoaffinity chromatography [VL Korol'kov et al., Lett. Pept. Sei, 2000, 7, 53-61], as well as in j # w-produced monoliths with a peptide ligand for immunoaffinity chromatography [R. Hahn et al., Anal. Chem., 73 (2001) 5126-5132].

Description of the preferred embodiments

The present invention provides cation exchange type enantioselective molecular recognition materials and cation pairing type enantioselective molecular recognition materials that are inorganic, organic, or based on an inorganic / organic hybrid, chiral chemical compounds or materials made of chiral synthons of low molecular weight and at least one free functional acid group with pK _a <4.0 (based on purely aqueous conditions) are constructed as synthetic chiral acid selectors of low molecular weight. The chiral acidic selectors preferably have the following additional structural features, preferably in combination:

Close proximity of the functional acid group to the nearest stereogenic center, which means that the acid function is either in the α, β or γ position to the stereogenic center,

Steric barriers which are bulky aliphatic, alicyclic or aromatic groups,

• aromatic groups with π-acidic or π-basic properties as π-π interaction sites.

The functionalized materials according to the invention are preferably constructed from a chiral acidic selector of low molecular weight and a polymer matrix which are connected to one another by means of a spacer or tether, wherein the functional acid group of the chiral acidic selector of low molecular weight can be selected from the group consisting of a sulfonic acid, Phosphonic acid, phosphinic acid, phosphoric acid, boric acid, a phosphonic acid monoamide, phosphoric acid monoamide, an amidosulfonic acid and others.

The chiral acidic selector of low molecular weight is preferably immobilized on a solid support which is selected from the group consisting of silicon dioxide (SiO ₂ ), aluminum oxide (Al ₂ O ₃ ), zirconium dioxide (ZrO ₂ ), titanium oxide (TiO ₂ ), by sol -Gel technology produced matrices, organic-inorganic, siliceous hybrid materials, optionally crosslinked polysiloxanes, any polymer obtained from vinyl monomers, optionally crosslinked poly (meth) acrylates, optionally crosslinked poly (meth) acrylamides, optionally crosslinked polystyrenes, mixed Styrene (meth) acrylate polymers, ring opening methathesis polymers, polysaccharides, agarose and one of the forms contained in the group comprising beads, monolithic or continuous materials, nanoparticles, membranes, resins and surface-limited layers.

The chiral acid selector can be incorporated into an inorganic, organic or inorganic / organic hybrid-like polymer which is obtained from a chiral functional acid monomer of low molecular weight, the functional monomer e.g. is a vinyl compound or an alkoxysilane and the polymer has one of the forms contained in the group comprising beads, monolithic or continuous materials, nanoparticles, membranes, resins and surface-limited layers.

The spacer, linker or tether is of any length and functionality or can have the general structure - (CH ₂ ) _n iY- (CH ₂ ) _nj - where Y is absent or is any of the functional groups (a) - (k) shown below and nj, nj are digits between 1 and 18.

(f) (9) (h) (i)

G) (k)

The chiral acid selector of low molecular weight can be selected from the group consisting of natural and unnatural amino acids, hydroxycarboxylic acids, aminophosphonic acids, aminophosphinic acids, aminosulfonic acids, hydroxysulfonic acids, Amidosulfonic acids, Aminoborsäuren, hydroxyphosphonic acids, Mercaptophosphonsäuren, Hydroxyphosphinsäuren, Amidophosphonsäuren, Phosphonsäuremonoamiden, Phosphorsäuremonoamiden, tartaric acid derivatives, mandelic acid derivatives Camphersulfonsäurederivaten, linear or cyclic, natural and non-natural peptides, linear or cyclic sulfo-, linear or cyclic Phosphonopeptiden or a compound consisting of at least one Components of this group is constructed.

The chiral synthon from which the low molecular weight chiral acidic selector is synthesized is taken from the group of chiral compounds specified above and matches any of the general structures (1) - (15) shown below, where X can be selected from the group consisting of phosphonic acid, sulfonic acid, phosphinic acid, boronic acid, phosphoric acid, amidophosphonic acid, amidosulfonic acid, amidophosphinic acid, where Y is the meaning of one of the groups set out above

(a) - (k) and where R, Ri R; one of the substituents from the group consisting of

Is hydrogen, optionally substituted aliphatic, aromatic, araliphatic, heteroaromatic and alicyclic substituents and the chiral acidic selector of low molecular weight is oligomerized, polymerized, immobilized, polymerized, composed, etc. via any of the substituents R, Ri Ri incorporating a spacer as set out above.

(10) (11) (12) (13)

The chiral low molecular weight acidic selector is preferably immobilized on thiol, amino, hydroxy, carboxy, epoxy, vinyl modified silicon dioxide, polymethacrylate polymer beads and resins, polyacrylamide polymer beads and resins, tentacle-like silicon dioxide or organic polymer beads and resins.

The spacer which binds the chiral acidic selector of low molecular weight to the optionally modified or activated carrier can be achieved by a radical addition reaction of a thiolalkyl-modified carrier to a vinyl group attached to the selector component or can be achieved by asymmetrically reacting a diisocyanate with an amino- or hydroxyalkyl-modified one Carrier and an amino- or hydroxy-modified selector component or by reacting an amino-, hydroxy- or thiol-modified carrier with a chlorine- or bromalkanoyl-derivatized selector, or by coupling an amino-modified carrier and an amino-modified selector by reacting one of the 2 constituents with a dicarboxylic anhydride. Spacer component and subsequent activation of the resulting carboxylic acid function and reaction with the second amino component or alkoxysilane with a terminal functionality for coupling to a selector component and subsequent binding of the resulting alkoxysilane-modified selector to silica gel or by any other suitable approach for a selector immobilization.

The above-mentioned X, Y and R groups or substituents can have the meaning set out below:

(1)

The X, Y and R groups or substituents can also have the meaning as set out below:

or

Y i Y, R. IR2 R3! -i! R5 In! R *! R: iRs Im 11 IR!

C = O CONH- H liBu | H 1 phenyl! H | 1 1 iBu | H ¹ phenyl! 1 jl 'tBu!

C = 0 i CONH- H iBu H! iBu i H il I phenyl j Hä iBu i 1 i 1 | tBu 1

The present invention further relates to the use of one of the functionalized materials described herein for the molecular recognition, binding and separation of chiral compounds, the chiral compound being selected from A group comprising basic drugs, basic synthons or intermediates, amino acids, peptides, proteins, nucleotides, aminoglycosides and other basic biomolecules, and methods used to form reversible molecular compound products and to separate mixtures of these compounds from the compounds with at least one of the compounds herein contact the functionalized materials described, the steps of contacting and separating using a. Liquid phase separation techniques, which include liquid chromatography, capillary electrophoresis, capillary melachrochromatography or supercritical liquid chromatography, or b. an extraction methodology, which includes liquid-liquid extraction, countercurrent liquid chromatography, centrifugal distribution chromatography, liquid-solid extraction technology, supported liquid membrane or fixed-site membrane technology.

Brief description of the drawings

1 schematically shows the structure of the chiral selector according to the invention, 1 denoting the chiral selector, 2 denoting chiral component, X denoting cation exchange group, 3 denoting spacer and 4 denoting carrier.

Examples

The present invention will now be described with reference to the following non-limiting examples.

Example 1: Synthesis of a weak chiral cation exchanger based on (S) -O-allyl-N- (3,5-dichlorobenzoyl) tyrosine, N-DCB-O-allyl-Tyr

(a) Synthesis of a cation exchange type N-Boc- (S) -tyrosine (3 mmol) (Propeptide, Vert Le Petit, France) was dissolved in 2 ml of dichloromethane and 2 ml of trifluoroacetic acid (Aldrich) and the reaction mixture was 3 hours long stirred at room temperature. After removal of the solvents under reduced pressure, (S) -O-allyl-tyrosine was obtained as a crude product which was used for the next step without further purification. 3,5-dichlorobenzoic acid N-hydroxysuccinimide ester was according to.

Standard reaction protocols were prepared from equimolar amounts of N-hydroxysuccinimide (Aldrich) and 3,5-dichlorobenzoic acid (98%) (Acros Organics, Geel, Belgium) in ethyl acetate in the presence of an equimolar amount of dicyclohexylcarbodiimide (Aldrich).

3 mmol (S) -O-allyl tyrosine and 15 mmol sodium bicarbonate were dissolved in 100 ml water. 3 mmol of 3,5-dichlorobenzoic acid N-hydroxysuccinimide ester were suspended in this solution with vigorous stirring. After 15 hours, the unreacted reagent was removed by filtration and the filtrate was acidified by dropwise addition of IM HC1. The precipitate thus formed was isolated by filtration and washed with water. The crude product was further purified by means of flash chromatography using silicon dioxide (chloroform-methanol as eluent with increasing volume fractions of methanol) (85% overall yield).

1H NMR (CDC1 ₃ ) δ 3.25 (q, 2H) 4.5 (d, 2H) 5.02 (q, 1H) 5.3 (d, 1H) 5.4 (d, 1H) 6.05 (m, 1H) 6.45 (d, 1H) 6.85 (d, 2H) 7.05 (d, 2H) 7.35 (s, 1H) 7.55 (s, 2H) ppm.

(b) Immobilization of a selector on silicon dioxide

Kromasil 100-3.5 μm (purchased from Eka Chemicals, Bohus, Sweden) was modified by refluxing in toluene with 3-mercaptopropyltrimethoxysilane (ABCR, Karlsruhe, Germany) (elemental analysis of the modified particles: 4.58% C, 1.0% H).

Then (S) -N-DCB-O-allyl-Tyr was covalently attached to this thiol-modified sorbent by a radical addition reaction following a standard procedure [M. Lämmerhofer and W. Lindner, J. Chromatogr. A, 741 (1996) 33]. An elemental analysis of the chirally modified silicon dioxide particles gave the following results: 15.19% C, 1.73% H, 0.64% N for CSP 1 with the N-DCB-O-allyl-Tyr selector (which means a medium SO- Occupancy of 0.44 mmol SO / g CSP corresponds).

Example !: Synthesis of a strong chiral cation exchanger based on (R) -N- (4-allyloxy-3,5-dichlorobenzoyl) -l-amino-3-methylbutanephosphonic acid, N- (4-allyloxy-DCB) -LeuP.

(a) Synthesis of a cation exchange type selector

For the synthesis of 4-allyloxy-3-5-dichlorobenzoic acid chloride, 9.7 mmol of 3,5-dichloro-4-hydroxybenzoic acid (Acros Organics, Geel, Belgium), 20 mmol of potassium hydroxide and 11.6 mmol of allyl bromide (Aldrich) were in for 24 hours Ethanol refluxed. After Hydrolysis of ester by-products with 1.8 M potassium hydroxide solution, the carboxylic acid was precipitated by acidification with 2M HC1. Recrystallization from petroleum ether-methanol gave the pure carboxylic acid in 92% yield. A 0.5 mmol aliquot was then refluxed with thionyl chloride for 3 hours, evaporated to dryness, and the remaining acid chloride was dissolved in dry dioxane for subsequent reaction with the aminophosphonic acid.

0.5 mmol (R) -l-amino-3-methylbutanephosphonic acid, the phosphonic acid analogue of L-leucine, L-LeuP (for synthesis see elsewhere [F. Hammerschmidt and F. Wuggenig, Tetrahedron Asymmetr., 10 (1999) 1709 ), was suspended in 10 ml of dry dioxane. After adding 1.5 mmol of N, O-bis-trimethylsilylacetamide (BSA, Aldrich), the mixture was heated at 70 ° C. for 3 hours until a clear solution was obtained. Thereafter, 0.5 mmol of N-ethyldiisopropylamine was added, followed by the dropwise addition of 0.5 mmol of 4-allyloxy-3,5-dichlorobenzoic acid chloride with ice cooling. The reaction mixture was allowed to slowly rise to room temperature. Evaporation of the solvent after 4 hours of stirring and the clean-up of the crude product by flash chromatography with silicon dioxide and chloroform-methanol eluent with increasing volume fractions of methanol gave the pure product in 50% yield.

1HNMR (DMSO) δ 0.85 (d, 6H) 1.4-1.7 (m, 3H) 4.3 (q, 1H) 4.6 (d, 2H) 5.3 (d, 1H) 5 , 45 (d, 1H) 6.1 (m, 1H) 8.0 (s, 2H) 8.45 (d, 1H)

(b) Immobilization of SO: same as in example lb

An elemental analysis of the chirally modified silicon dioxide particles (see FIG. 1) gave the following results: 8.02% C, 1.25% H, 0.3% N for CSP 2 with the N- (4-

Allyloxy-DCB) -LeuP selector (which corresponds to an SO occupancy of 0.21 mmol SO / g CSP).

Example 3: Use of materials from Examples 1 and 2 for the enantiomer separation of chiral bases:

(a) Comparison of strong versus weak chiral cation exchangers It was assumed that the replacement of the carboxylic acid function of the selector by a phosphonic acid group as the primary ionic interaction site was favorable with regard to SO-S A binding enthalpies and enantioselectivities. Non-Aqueous Cation Exchange Capillary Electrochromatography (NA-CEC) Data Shown in Table 1 confirm the higher enantioselectivity of the strong cation exchanger (CSP 2, material from Example 2, based on an N- (4-allyloxy-DCB) -LeuP selector) compared to the weak counterpart (CSP 1, material from Example 1, based on ( S) -N-DCB-O-allyl-Tyr).

Table 1: Enantioselectivities of some basic analytes on CSP 1 and CSP 2. ^a

CSP 1

Analytes t__ (min) tR ₂ (min) α ^b R _s Ni n ¹ ) N ₂ (m- ¹ )

Quinine / Quinidine 13.19 15.12 1.21 3.70 45900 49400 Mefloquine 13.40 13.85 1.05 1.01 65400 59700

Metoprolol 11.10 1.00 - 37500

Atenolol 14.41 1.00 _ 44000

CSP 2

Analytes ti (min) tw> (min) α "R _s Ntfm ¹ ) l__L_ι -_is

Quinine / Quinidine 13.48 15.37 1.23 4.53 62800 93400

Mefloquine 22.49 25.07 1.15 3.16 61 600 49000

Metoprolol 12.83 13.31 1.06 1.39 81 500 100 400

Atenolol 19.00 19.67 1.05 1.33 94 400 95 300 ^a Column dimension: 250 (335) x 0.1 mm id, mobile phase: 50 mM 2-aminobutanol and 12 mM HCOOH in

ACN - MeOH (80/20, vol.%); T: 20 ° C; Voltage: + 15 kV; Injection: +5 kV for 5 s ^b o = k _app2 / k _{app ,;} k _app = (t _R -to) / to; to (CSP 1) = 3.91 min; t ₀ (CSP 2) = 4.90 me l

Table 1 clearly shows that the more acidic cation exchanger gives considerably better results.

(b) Use of a strong chiral cation exchanger based on an N- (4-allyloxy-DCB) -LeuP selector (CSP 2) for the enantiomer separation of different β-blockers by non-aqueous capillary electrochromatography (NA-CEC)

Table 2: NA-CEC enantiomer separations from different ß-blockers on CSP 2. ^a

Talinolol 25.16 26.37 1.06 1.68 80800 82000 Analytes t _R ι (min) t∞ (min) R «Nifm- ¹ ) N dn ^'1 )

Bupranolol 13.19 12.59 1.05 1.21 90200 117500

Bunitrolol 11.88 12.27 1.06 1.11 58000 91400

Celiprolol 14.01 14.44 1.05 1.20 97000 99500

Penbutolol 15.09 15.67 1.06 1.46 94000 98000 tert-butyl-atenolol 19.14 20.07 1.06 1.75 90 200 85 400 tert-butyl-metoprolol 12.96 13.58 1.07 2, 02 119300 126000 tert-butyl propanolol 14.88 15.60 1.07 1.95 110100 105100

Atenolol ^b 19.00 19.67 1.05 1.33 94 400 95 300

Metoprolol 12.84 13.31 1.06 1.39 81 500 100 400

Propranolol 15.12 15.44 1.03 0.67 58100 67600

Acebutolol 13.17 13.47 1.03 0.7 64 200 63 200

Practolol 16.85 17.44 1.05 1.30 91400 93200

Alprenolol 11.79 12.00 1.03 0.63 78000 90600

Pindolol ⁰ 15.99 16.39 1.04 0.95 88300 101400

Mepindolol 15.27 15.58 1.03 0.68 82200 73000

Metipranolol 11.34 11.56 1.03 0.59 53400 69200

Normetoprolol 5.59 5.73 1.16 0.61 65200 29600

Ethoxymethyl-11.67 12.07 1.06 1.40 105200 116300

metoprolol

Ethoxyethyl-11.82 12.23 1.06 1.25 84000 96800

metoprolol

4-methoxy-14.38 14.62 1.02 0.62 108600 76100

propranolol

4-hydroxy- 24.22 24.54 1.02 0.42 - -

propranolol

O-allyl propranolol 11.15 11.43 1.04 0.96 84000 115400

Oxprenolol 11.38 11.65 1.04 0.79 52200 106200

Carazolol 16.88 17.15 1.02 0.59 126400 73400

Sotalol 13.92 14.44 1.06 1.49 100500 108200

Nifenalol 10.41 10.88 1.09 1.11 33400 53100 ^a conditions see table 2, t ₀ (acetone) = 4.9 min ^b first eluted enantiomer: (S)

⁰ first eluted enantiomer: (-) ^d eluent: 50 mM 2-aminobutanol and 4 mM HCOOH in ACN-MeOH (80:20, vol%); t ₀

(Acetone) = 4.78 min (c) Use of a strong chiral cation exchanger based on an N- (4-allyloxy-DCB) -LeuP selector (CSP 2) for the enantiomer separation of different basic drugs by non-aqueous capillary electrochromatography (NA-CEC)

Table 3: NA-CEC enantio separations from various basic analytes on CSP 2. ^a

Analytes t _W ι (min) tτn (min) α R <Nidn ¹ ) N m- ^{1 "} *

Propafenone 11.34 11.58 1.04 0.64 55600 62400

Norpropafenon 5.46 5.57 1.11 0.73 82400 99800

Bamethane ^b 25.28 26.33 1.05 1.05 51000 37500

Ephedrine 26.16 26.42 1.01 0.41 - -

Mefloquine 22.49 25.07 1.15 3.16 61 600 49000

Quinidine / quinine ^b 13.48 15.37 1.23 4.53 62800 93500

Oxyphencyclimin 20.61 21.59 1.06 2.15 134700 138400

Etidocaine ⁰ 16.13 16.49 1.03 1.51 254000 360200

Bupivacaine ^d 14.18 14.42 1.03 0.9 192000 187800

Tröger's Base ^d 6.59 6.70 1.08 0.77 150,000 100,000

Promethazine ^d 19.80 20.19 1.03 1.46 361000 375800

Dixyrazine 20.29 20.46 1.01 0.38 - -

Pheniramine ^d 13.58 13.82 1.03 0.58 103800 50800

Doxylamine ^d 13.18 14.42 1.16 0.79 3900 14200

Benzetimide ⁶ 8.25 8.36 1.04 0.52 192600 65000

Camylofin ⁰ 11.86 12.17 1.05 0.48 40,000 14,000

Lorazepam ^f 7.60 7.79 1.08 0.7 58600 50600

O - (/ ert-butyl- 6.78 9.34 2.21 15.28 85200 53400 carbamoyl) -

Mefloαuin ^{f a} voltage: +15 kV, electrolytes: see footnote bf, for other conditions see Table 2; ^b 50 mM 2-aminobutanol, 12 mM HCOOH; t ₀ (acetone) = 4.90 min ^c 10 mM TEOA, 100 mM HCOOH; t ₀ (acetone) = 4.99 min ^d 10 mM 2-aminobutanol, 100 mM HCOOH; t ₀ (acetone) = 5.41 min ^e 10 mM 2-aminobutanol, 2.4 mM HCOOH; t ₀ (acetone) = 5.71 min ^f 50 mM TEOA, 5 mM AcOH; Voltage: + 25 kV; t ₀ (acetone) = 5.32 min Tables 2 and 3 clearly illustrate the broad range of applications of the cation exchangers according to the invention for chiral bases of various types.

Example 4: Synthesis of a strong chiral cation exchanger based on (R) -N- (4-allyloxy-3,5-dichlorobenzoyl) -2-amino-3,3-dimethylbutanesulfonic acid

(a) Synthesis of the selector:

The chiral selector based on sulfonic acid was synthesized according to the procedure described in Example 2a by N-acylation of (R) -2-amino-3,3-dimethylbutanesulfonic acid, using 4-allyloxy-3,5-dichlorobenzoic acid chloride. After evaporation of the solvent, the product was purified by preparative chromatography on a chiral stationary phase based on tert-butylcarbamoylquinidine (d _p = 15 μm; column dimension: 250 x 16 mm ID; eluent: methanol - 1.5 M ammonium acetate (80:20, Vol.%) (PH _a = 6.0); flow rate: 6 ml / min, room temperature) isolated from the crude reaction mixture. The pure fraction of the chromatographic run was desalted on a cation exchanger (Dowex 50W, water as eluent). The eluate was evaporated to give the pure product.

1H NMR (D ₂ O) δ 7.6 (s, 2H), 6.0 (m, 1H), 5.25 (d, 1H), 5.1 (d, 1H), 4.45 (d, 2H), 4.1 (d, 1H), 2.7-3.2 (m, 2H), 0.75 (s, 9H) ppm.

(b) Immobilization of SO on thiol-modified Kromasil 100-3.5 μm:

A procedure similar to that described in Example 1b was used, but a water-methanol mixture (80:20, vol.%) Was used as the solvent and 2,2'-azobis (2-amidinopropane) dihydrochloride as the initiator.

100 mg selector was dissolved in 3 ml of a water-methanol mixture (80:20, vol.%). 200 mg of 3-thiolpropyl-modified Kromasil 100-3.5 µm were suspended in this solution, and 10 mg of water-soluble initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride, was added. The mixture was sonicated for 15 minutes and flushed with N _{2 for} 10 minutes. The radical addition reaction was allowed to continue at 50 ° C overnight (24 h). The modified silicon dioxide was then isolated and washed with various solvents. An elemental analysis of the chirally modified silicon dioxide particles gave the following results: 10.06% C, 1.62% H (which corresponds to an SO occupancy of 0.29 mmol SO / g CSP).

(c) Test chromatogram: separation of clenbuterol enantiomers

Separation of the enantiomers of clenbuterol on a strong chiral cation exchanger based on ß-aminosulfonic acid by NA-CEC. Column dimensions: 250 x 0.1 mm ID (L _ef f = 250 mm, L _to = 335 mm), eluent: 50 mmol / L formic acid and 25 mmol / L 2-aminobutanol in acetonitrile - methanol (80:20, vol. %); Voltage: + 15 kV (18.2 μA); Temperature: 20 ° C.

(d) Electrochromatographic results:

Table 4: Separation of enantiomers from different chiral bases by NA-CEC

Versuchsbed. : CSP: (S) -N- (4-allyloxy-3,5-dichlorobenzoyl) -2-amino-3,3-dimethylbutanesulfonic acid, which is immobilized on 3-mercaptopropyl-modified Kromasil 100 3.5 μm; Capillary _dimensions : L _tot 33.5 cm, L _eff 25.0 cm, L _packec j 25.0 cm; Eluent: 25 mM 2-amino-1-butanol and 50 mM formic acid in acetonitrile-methanol (80:20, vol.%); Voltage: + 15 kV; T: 20 ° C

Ephedrine 25.84 27.60 1.10 3.64 47176 50190 ()

Nifenalol 40.64 48.03 1.23 8.37 51065 33970 -

Bamethane 34.26 35.25 1.04 1.69 57759 56195 -

Sotalol 32.06 33.46 1.06 2.59 57999 59337 (-)

Atenolol 41.67 42.24 1.02 0.7 39748 44487 - tert-butyl-atenolol 38.87 40.15 1.04 1.42 50618 51427 -

Propafenone 21.93 22.78 1.06 2.05 45393 47245 (S)

Talinolol 63.70 65.93 1.04 1.62 36951 34133 -

Bupranolol 23.99 24.93 1.06 222 52401 55085 -

Celiprolot 30.78 32.44 1.07 2.8 45394 45815 -

Bunitrolol 24.44 25.65 1.08 2.67 48931 48769 -

Practolol 41.42 41.86 1.01 0.59 52792 49S45 -

Oxprenolol 17.93 18.28 1.04 0.96 31 350 52 185 -

Metipranolol 21.91 22.31 1.03 0.95 41433 45335 -

Alprenolol 23.22 23.78 1.04 1.37 48425 56473 -

Penbutolol 23, ^l 32 24.33 1.07 1.59 22402 21938 (S)

Isopenbutolol 24.93 25.91 1.06 2.18 50075 52309 (S)

[≤operϊbutolol 27.80 29.17 1.07 2.63 49773 47004 (S)

Carazolol 36.48 37.20 1.03 1.13 50456 55998 -

Pindolol 26.12 26.65 1.03 1.07 40 194 54 403 -

Mepindolol 26.62 27.16 1.03 1.17 54292 55592 -

Acebutolol 32.72 33.91 1.05 2.03 50314 53015 -

Propranolol 29.19 29.70 1.03 0.97 50181 49762 - tert-Butyi-Propranolol 31.55 33.02 1.06 2.6 50639 54365 -

4-methoxy-propranolol 32.47 33.10 1.03 1.08 49874 52499 - tert-butyl-propranolol 31.87 33.25 1.06 2.41 51663 51952 -

O-allyl propranolol 18.54 18.98 1.04 1.33 48899 55906 -

4-hydroxy-propranolol 40.21 41.15 1.03 1.26 44240 51540 (S)

Metoprolol 25.17 25.47 1.02 0.63 41944 45582 - teπ-butyl-metoprolol 22.29 22.94 1.04 U21 31978 25712 -

E tho xymethy lmeto- 25 ^ 04 25.24 1.01 0.47 56982 50222 - prolol

Ethoxyethylmetoprolol 24.19 24.40 1.01 0.5 56 136 54812 -

Benzetimide 23.83 25.43 1.10 3.43 44234 45678 (R)

Etidocaine 11.63 11.87 1.07 0.86 37017 21999 -

Mefloquine (MQ) 69.16 89.49 1.33 12.02 35279 35366 -

0- (tert- 33.45 48.98 1.62 29.99 36271 347589 -

Butylcarbamoyl) -MQ

Pantoprazole 10.87 11.34 16 1.77 27833 27272 -

Omeprazole 13.29 14.29 1.20 2.51 22914 16727 «

N-Deisopropyl-20.45 21.05 1.05 1.23 23600 38233 _ disopyramide

Disopyramide 9.30 9.46 1.14 0.51 8199 30576 Phenmerazine 41.10 43.44 1.07 2.59 39513 31802 _

Sulpirid 15.71 16.14 1.05 0.96 15706 28631 »

1 - (2.4-dichlorophenyl) - 14.66 15.12 1.07 0.84 6212 28440.

2- (l-imidazolyl) ethanol Connection tRI tR. α Rs Nι N- eo ^{, l}

Tryptophanamide 49.54 51.94 1.06 0.71 3247 4054

Tramadol 10.82 11.14 ι, π 1.03 15394 26872

Metanephrine 31.34 32.01 1.03 1.18 48333 52128

1 - (_ l-Naphtyl) ethylamine 41.10 42.93 1.06 2.56 29837 140023 - Pronethacfol 31.83 34.91 1.13 5.4 53692 56179

Buticide 10.71 10.88 1.06 0.65 28546 26692

Isoxsuprine 24.93 27.90 1.18 5.91 41 895 46861 (-r)

Miconazole 12.03 12.35 1.07 0.77 14667 13197

Dipivefrin 25.99 26.92 1.05 1.98 49295 52467

Terfenadine 26.47 26.82 1.02 0.64 34893 40932

Eprazinon 10.28 10.57 1.10 1.15 25619 27507

Salbutamol 42.27 47.28 1.14 6.82 94682 42474

Orciprenaline 52.42 54.27 1.04 1.71 10462 37463

Terbutaline 52, '77 57.79 1.11 6.2 80914 70211

Clenbuterol 36.43 45.92 1.33 14.18 58876 62213

Mebeverin 11.38 11.78 1.13 0.97 9955 15880

Hydroxyzine ^" 15.35 15.55 1.03 0.58 25654 35311

Flecainide 18.44 18.93 1.05 1.39 43394 45500

Flupentixol 25.52 28.30 1.15 5.77 50729 49296

Rimiterol 55.13 59.77 1.10 2.77 19330 18537

Oxyphencyclimin 10.19 10.36 1.08 0.57 12765 28711 ^a stereochemical descriptor (absolute configuration, optical rotation) of the first eluted enantiomer

The results of Table 4 provide strong evidence of the extremely broad range of uses of the enantioselective strong cation exchangers of the present invention. The invented separation principle thus represents a tool that can be regarded as generally applicable to the enantiomer separation of chiral basic compounds and - as illustrated by the example of tryptophanamide - also for amino acids and peptides.

Example 5: Comparison of the chromatographic behavior of on β-aminocarbon,

Weak and strong chiral based on phosphonic and sulfonic acids

cation

CSP 3: X = COOH, R! = R ₂ = H, R ₃ = tert-butyl, (S) configuration

CSP 4: X = SO ₃ H, Ri = R ₂ = H, R ₃ = tert-butyl, (S) configuration

CSP 5: X = SO ₃ H, Ri = R ₂ = H, R ₃ = COOH, (R) configuration

CSP 6: X = SO3H, Ri = R ₂ = CH ₃ , R ₃ = COOH

CSP 7: X = PO ₃ H ₂ , Ri = R ₂ = H, R ₃ = tert-butyl

* denotes a stereogenic center that is either R or S.

Table 5: NA-CEC enantiomer separations of various basic compounds. ¹

CSP 3 CSP 4 CSP5

Analytes α R, R «oc R <

Mefloquine (MQ) 1.10 4.87 1.29 12.02

MQ tert-butyl 1.13 4.25 1.46 29.99 carbamate

Ephedrine 1.00 0.00 1.07 3.64 1.01 0.20

Sotalol 1.00 0.00 1.04 2.59 1.01 0.60

Propafenone 1.00 0.00 1.04 2.05 1.00 0.00

Rimiterol 1.00 0.00 1.08 2.77 1.01 0.32

Bunitrolol 1.00 0.00 1.05 2.67 1.00 0.00

Benzetimide 1.02 0.64 1.07 3.43 1.00 0.00

Clenbuterol 1.03 1.34 1.26 14.18 1.03 1.82

Nifenalol 1.05 2.40 1.18 8.37 1.02 0.95 ^a column dimension: 250 x 0.1 mm ID (L _e ff = 250 mm, L _to t = 335 mm), eluent: 50 mmol / L formic acid and 25 mmol / L 2 -Aminobutanol in acetonitrile - methanol (80:20, vol.%); Voltage: + 15 kV; Temperature: 20 ° C

The direct comparison of carbon (CSP 3) and sulfonic acid counterparts (CSP 4) (Table 5) clearly shows the significantly increased ability to enantiotere the stronger cation exchanger, as by the resolution values (R _s ), which are higher in all test samples for CSP 4 , is displayed.

Example 6: Synthesis of a peptide-derived selector or CSP

(a) Oxidation of (Cys-Leu) ₂ to the corresponding sulfonic acid dipeptide

A performic acid solution is freshly prepared by mixing 0.5 ml H ₂ O ₂ (30% in water) with 9.5 ml formic acid for 2 hours at room temperature. The oxidized dimeric (Cys-Leu) ₂ dipeptide is dissolved in formic acid-methanol (5: 1, vol.%) (40 mg / ml). The 2 solutions are cooled to 0 ° C, and then the peptide solution is added dropwise to the performic acid. The solution is then stirred at 0 ° C for 2.5 hours. To stop the reaction, ice is added to the reaction mixture. The solvent is then removed by evaporation.

(b) Acylation with 4-allyloxy-3,5-dichlorobenzoic acid hydroxysuccinimide ester

The 0.5 mmol of the sulfonic acid dipeptide was dissolved in 25 ml of water with vigorous stirring. 7 mmol of NaHCO ₃ dissolved in 10 ml of water and 0.7 mmol of 4-allyloxy-3,5-dichlorobenzoic acid hydroxysuccinimide ester were added successively. The reaction mixture was stirred vigorously at room temperature for 16 hours. After evaporation of the solvent, the product was purified by preparative chromatography on a chiral stationary phase based on tert-butylcarbamoylquinidine (d _p = 15 μm; column dimensions: 250 x 16 mm ID; eluent: methanol - 1.5 M ammonium acetate (80:20, Vol.%) (ΡH _a = 6.0); flow rate: 6 ml / min, room temperature) isolated from the crude reaction mixture. The pure fraction of the chromatographic run, which contained the product, was evaporated to dryness, dissolved in water and desalted on a cation exchanger (Dowex 50W, water as eluent). The eluate was evaporated to dryness to give the pure N- (4-allyloxy-3,5-dichlorobenzoyl) - CySO ₃ H-Leu dipeptide product.

(b) Immobilization of SO on thiol-modified Kromasil 100-3.5 μm: see example 4b.

Example 7: Determination of pK _a values (based on aqueous conditions, pK _{a aq} )

(a) under aqueous conditions 1 mmol acid is dissolved in 100 ml water and by potentiometric titration with

0.1 mol / 1 NaOH solution titrated. The pK is determined by fitting the titration curve to the experimental data, which makes the pK for the curve with the best

Adjustment results.

(b) under mixed aqueous-organic conditions pK _a - values of selectors which are not soluble under purely aqueous conditions were determined at 24 ° C. using a Sirius titrator from Sirius Analytical Instruments Ltd (East Sussex, UK) in various methanol Water ratios titrated. The pK _a values with the

Values from Example 7a are comparable, were then using the Yasuda

Shedlovsky operation by back extrapolation to zero percent organic

Obtain auxiliary solvents.

Example 8: Synthesis of chiral organic cation exchange polymer beads

(a) Thiol modified polymethacrylate beads

10 g of poly (glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads (Suprema lOOOu) were suspended in 100 ml of methanol and sonicated for 10 minutes to break all aggregates and to obtain a homogeneous slurry. The slurry was transferred to a flask equipped with a mechanical stirrer. 80 mmol (= 9.4 ml) 1,4-butanedithiol and 80 mmol (= 4.48 g) potassium hydroxide, which was dissolved in 30 ml methanol, were added. The reaction mixture was stirred under a nitrogen atmosphere at room temperature for 72 hours. The polymer particles were then filtered, washed thoroughly with methanol, THF, chloroform and diethyl ether (twice each) and dried. An elemental analysis showed 8.0% (wt.%) S, which corresponds to an occupancy of 1.25 mmol thiol / g polymer.

(b) Immobilization of (R) -N- (4-allyloxy-3,5-dichlorobenzoyl) -2-amino-3,3-dimethylbutanesulfonic acid on thiol-modified organic polymer beads

0.5 g of thiol-modified polymer particles were suspended in 25 ml of a water-methanol mixture (80:20, vol.%), Sonicated for 10 minutes and transferred to a flask equipped with a mechanical stirrer and a condensate cooler. 300 mg (R) -N- (4-allyloxy-3,5-dichlorobenzoyl) -2-amino-3,3-dimethylbutanesulfonic acid and 30 mg of 2,2'-azobis (2-amidinopropane) dihydrochloride as an initiator were added. The reaction mixture was refluxed under a nitrogen atmosphere for 18 hours. The particles were washed thoroughly with water-methanol, methanol, chloroform, diethyl ether and dried. An elemental analysis showed a selector coverage of 102 μmol / g polymer.

Claims

claims:

1. enantioselective cation exchange material, comprising a chiral selector (1), which is composed of a chiral component (2) and at least one cation exchange group (X), a spacer (3) and a support (4),

characterized in that

the chiral component (2) has a molecular weight of less than 1,000 and the at least one cation exchange group (X) is an acid group with a pKa <4.0.

2. Enantioselective cation exchange material according to claim 1, characterized in that the acid group has a pKa <3.5.

3. Enantioselective cation exchange material according to claim 2, characterized in that the acid group has a pKa <2.5.

4. enantioselective cation exchange material according to any one of claims 1-3, characterized in that the acid group is a sulfone, sulfine, phosphorus, phosphone or phosphine group.